13. A composition comprising the composition of claim 1, a therapeutic
agent and a pharmaceutically acceptable carrier.

14. The composition of claim 13, wherein the therapeutic agent is an
antineoplastic therapeutic agent.

15. The composition of claim 13, wherein the therapeutic agent is an
antiretroviral therapeutic agent.

16. The pharmaceutical composition of claim 13, wherein said therapeutic
agent is an antigen capable of stimulating an immune response.

17. A method for treating a condition linked to a hyperproliferative
disorder, comprising administering to a patient in need thereof an
effective amount of a composition according to claim 14.

18. A method of stimulating an immune response comprising administering
the composition of claim 16 to a patient.

Description:

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority under 35 U.S.C. §119(e) to
U.S. Provisional Patent Application Ser. No. 61/413,352, filed on Nov.
12, 2010, the disclosure of which is incorporated herein by reference in
its entirety.

FIELD OF THE INVENTION

[0003] The present invention relates to the synthesis of novel
polyethylene glycol-based (PEGTide) dendrons, a carrier for the in vivo
delivery of a therapeutic agent, compositions thereof and methods of use.
In particular, this invention is directed to the PEGtide dendron composed
from polyethylene glycol (PEG) and amino acids, as a carrier for the in
vivo delivery of a therapeutic agent. The invention comprises a dendron
structure, a tree like structure, similar in structure to a dendrimer,
consisting of peptide subunits interspersed with short monodisperse
poly(ethylene glycol)/PEG subunits. The PEG subunits endow dendrons with
favorable pharmaceutical (e.g., long circulation time, reduced toxicity,
increased water solubility etc.) and pharmacological (e.g.,
biocompatibility, reduced immunogenicity, biodistribution etc.)
properties, generally known to be associated with PEG. PEGtide dendrons
are useful for various applications including drug delivery and
diagnostic purposes because they can be modified with therapeutic,
targeting, biologic, and diagnostic agents.

[0005] Dendrimers (Greek: dendri: "tree-like" and meros: "part of") are
monodisperse macromolecules with well-defined branched architecture and
symmetrical morphology (Bosman, A. W., Chemical Reviews 99, 1665-1688,
(1999)). Dendrimers are comprised of a series of branches extending
outward from an inner core. These branches are arranged in layers, called
generations, and represent the repeating units (monomer) of a dendrimer.
A typical dendrimer molecule contains an inner core, layers of repeating
units, and multiple terminal functional groups. The active moieties are
either encapsulated into the core/cavities or grafted onto the surface of
dendrimers. The dendrimers are distinct from other nanocarriers in that
they possess a tunable structure, empty intramolecular cavity, and
multifunctional surface (Duncan, R., Advanced Drug Delivery Reviews 57,
2215-2237, (2005)). The existence of interior void spaces inside the
dendrimer, particularly the high generation dendrimers, becomes
appropriate locations for drug or gene material loading and
transportation. Generally, payloads are held inside the dendrimer
Cavities via covalent attachment, hydrophobic interaction, hydrogen
bonds, or charge interaction. The degradable or cleavable bonds like
ester, imine, acetal and ketal are more preferred in covalent bonding
since they function as the triggers of environmental response release.

[0007] Although dendrimers have found wide application in drug and gene
delivery, and diagnostics, their use is restricted due to
reticuloendothelial system (RES) uptake, immunogenicity, hemolytic
toxicity, cytotoxicity, hydrophobicity.

[0008] Statistics on dendrimer PEGylation shows that most of the studies
have focused on dendrimer surface modification. The toxicity from
dendrimer branch/core structure is still a potential threat for safe use
of dendrimers. In addition, since the sizes of currently developed
dendrimers are less than 100 nanometers, these are prone to excretion by
kidney.

SUMMARY OF THE INVENTION

[0009] The present invention relates to the synthesis of novel
polyethylene glycol-based (PEGTide) dendrons, a carrier for the in vivo
delivery of a therapeutic agent, and its application in drug delivery,
diagnostic applications, vaccines and related methods to treat a
condition, and generate an immune response.

[0010] Developing novel dendrons with larger size (100 to 200 nm) is
needed in order to meet the advanced requirements of drug delivery
applications in the pharmaceutical field, including better
biocompatibility, higher size, and tunable interior void size/structure,
design. The development of a PEGtide dendron is disclosed in this patent.

[0011] An embodiment of the present invention is a carrier for the in vivo
delivery of a therapeutic agent represented by Formula 1:

##STR00001##

[0012] wherein R is:

##STR00002##

[0013] R1 is:

##STR00003##

[0014] R1 is:

##STR00004##

[0015] PEG is linear or branched poly(ethylene glycol);

[0016] AA, AA1, AA2, and AA3 are each independently lysine
or ornithine;

[0021] In certain embodiments, Formula (I) may not contain PEG. In certain
other embodiments, R may not contain PEG, and Formula (I) may be
represented wherein R is:

##STR00006##

[0022] In certain embodiments, R may not contain Ld or Le. In
certain embodiments, R1 may not contain PEG. In certain embodiments,
R1 may not contain Lg or Lh. In certain embodiments,
R2 may not contain PEG. In certain embodiments, R2 may not
contain Lk or Lm.

[0027] In accordance with any of the above embodiments, the invention
further comprises at least one therapeutic agent. In a further
embodiment, a therapeutic agent may be a pharmaceutically active,
diagnostic, biologic, imaging, targeting agent or an adjuvant.

[0028] In accordance with any of the above embodiments, the invention may
further comprise at least one therapeutic agent and a pharmaceutically
acceptable carrier. In a further embodiment, a therapeutic agent may be
an antineopastic or an antiretroviral agent.

[0029] In accordance with any of the above embodiments, the invention may
be a vaccine comprising a composition represented by Formula 1.

[0030] The present invention further provides a method for treating a
condition linked to a hyperproliferative disorder, comprising
administering to a patient in need thereof an effective amount of a
composition represented by Formula 1 further comprising an antineoplastic
agent.

[0031] The present invention also provides a method for treating
stimulating an immune response, comprising administering to a patient in
need thereof an effective amount of a vaccine comprising a composition
represented by Formula 1 further comprising an antigen.

[0050] The instant invention relates to polyethylene glycol-based
dendrons, otherwise known as PEGtide dendrons, a carrier for the in vivo
delivery of a therapeutic agent, compositions thereof and methods of use.
PEGTide dendrons are synthesized with tunable nanostructure by regulating
size, shape, surface chemistry, and interior void space. Current
applications for commercial dendrons, or their surface-modified products,
include drug/gene delivery and/or imaging. With the increase of
generation in dendrimers like PAMAM or PEI dendrimers, positive charges
increase and therefore the toxicity of the carrier also increases. This
is not the case with PEGtide dendrons because the dendritic tree like
structure is interspersed with biocompatible PEG subunits. In order to
improve the biocompatibility and enlarge the application area, novel
polyethylene glycol-based dendrons (PEGTide) are designed and
synthesized.

2. Definitions

[0051] As used above, and throughout the description of the invention, the
following terms, unless otherwise indicated, shall be understood to have
the following meanings:

[0052] As used herein, the singular forms "a," "an" and "the" include
plural references unless the content clearly dictates otherwise.

[0053] The term "about", as used here, refers to +/-10% of a value.

[0054] As used herein, the term "linker" refers to a chemical moiety that
connects a molecule to another molecule, covalently links separate parts
of a molecule or separate molecules. The linker provides spacing between
the two molecules or moieties such that they are able to function in
their intended manner. Examples of linking groups include peptide
linkers, enzyme sensitive peptide linkers/linkers, self-immolative
linkers, acid sensitive linkers, multifunctional organic linking agents,
bifunctional inorganic crosslinking agents and other linkers known in the
art. The linker may be stable or degradable/cleavable.

[0055] As used herein the term "therapeutic agent" encompasses
pharmaceutically active therapeutic agents, diagnostic, biologic and
targeting agents, as well as adjuvants.

[0056] As used herein, the term "diagnostic agent" refers to any molecule
which produces, or can be induced to produce, a detectable signal. The
diagnostic agent may be any diagnostically useful compound that may be
bound via a functional group thereon to the composition of the invention.
Diagnostic moieties having reporter molecules that can be detected by
imaging equipment may include radioactive, paramagnetic, fluorescent or
radioopaque chemical entities. Non-limiting examples of labels include
radioactive isotopes, enzymes, enzyme fragments, enzyme substrates,
enzyme inhibitors, coenzymes, catalysts, fluorophores, dyes,
chemiluminescers, luminescers or sensitizers; a non-magnetic or magnetic
particle, iodinated sugars used as radioopaque agents, and can be
appended to linker backbones using ester or other linkages.

[0058] As used herein the term "nucleic acid" refers to a molecular entity
composed of a nucleobase, sugar moiety, and phosphate group, or analogs
thereof, including DNA, RNAs, microRNAs, siRNAs, shRNAs, and tRNAs.
Examples include the DNA nucleotides, i.e., adenine, guanine, cytosine,
and thymidine, or the RNA nucleotide uracil, or synthetic analogs
thereof. Examples of sugar moieties to which the nucleobases are
covalently bonded include but are not limited to ribose and deoxyribose.
Analogs of sugars can also be present; for example, halodeoxyribose
analogs.

[0059] As used herein the term "peptide" is used interchangeably with the
term "protein" and "amino acid sequence", in its broadest sense refers to
a compound of two or more subunit amino acids, amino acid analogs or
peptidomimetics.

[0060] As used herein, the term "amino acid" refers to natural and/or
unnatural or synthetic amino acids, including glycine and both the D and
L optical isomers, amino acid analogs (for example norleucine is an
analog of leucine) and peptidomimetics.

[0061] As used herein, "targeting agents" refer to ligands, polymers,
proteins, cytokines, chemokines, peptides, nucleic acids, lipids,
saccharides or polysaccharides, small molecules or any combination
thereof, (for example a gylcolipid, glycoprotein etc) that bind to a
receptor or other molecule on the surface of a targeted cell. An
exemplary small-molecule targeting compound is folate, which targets the
folate receptor. The degree of specificity can be modulated through the
selection of the targeting molecule. For example, antibodies are very
specific. These can be polyclonal, monoclonal, fragments, recombinant, or
single chain, many of which are commercially available or readily
obtained using standard techniques. Examples of antibodies include, but
not limited to abciximab, basiliximab, cetuximab, infliximab, rituximab,
trastuzumab etc.

[0063] As used herein, the term "dendron" encompasses polymers
distinguished by their repeated branching structure emanating from a
central core. The term dendron also encompasses dendrimers. Preferably
the polymer is monodisperse PEG. Biocompatible PEG may be present
throughout the dendritic structure, and is not restricted to the core or
surface:

[0064] As used herein, the terms "polymer," "polymeric" and similar terms
have the usual meaning known to those skilled in the art and thus may be
used to refer to homopolymers, copolymers (e.g., random copolymer,
alternating copolymer, block copolymer, graft copolymer) and mixtures
thereof.

[0065] As used herein, "PEG", is used herein as an abbreviation for
polyethylene glycol. PEGs are included within the broader class of
polyalkylene oxides, which include PEG as well as polypropylene glycols,
and polyglycol copolymers. PEG can have a range of molecular weights. The
PEG molecular weight range contemplated for use in the present invention
is from about 100 to about 1000 Da.

[0066] "Animal" includes all vertebrate animals including humans. In
particular, the term "vertebrate animal" includes, but not limited to,
mammals, humans, canines (e.g., dogs), felines (e.g., cats); equines
(e.g., horses), bovines (e.g., cattle), porcine (e.g., pigs), mice,
rabbits, goats, as well as in avians. The term "avian" refers to any
species or subspecies of the taxonomic class ava, such as, but not
limited to, chickens (breeders, broilers and layers), turkeys, ducks,
geese, quail, pheasants, parrots, finches, hawks, crows and ratites
including ostrich, emu and cassowary.

[0067] The term "pharmaceutical composition" refers to the combination of
an active therapeutic agent with a carrier, inert or active, making the
composition especially suitable for diagnostic or therapeutic use in vivo
or ex vivo.

[0068] A "pharmaceutically acceptable carrier," after administered to or
upon a subject, does not cause undesirable physiological effects.

[0069] The term "immunogenic" refers to a capability of producing an
immune response in a host animal against an antigen or antigens.

[0070] "Immune response" refers to a response elicited in an animal, which
may refer to cellular immunity, humoral immunity or both. This immune
response forms the basis of the protective immunity elicited by a vaccine
against a specific infectious organism.

[0071] "Antigenic agent," "antigen," or "immunogen" means a substance that
induces a specific immune response in a host animal. It can be a molecule
containing one or more epitopes (either linear, conformational or both)
that elicit an immunological response. The term "epitope" refers to basic
element or smallest unit of recognition by an individual antibody, B-cell
receptor, or T-cell receptor, and thus the particular domain, region or
molecular structure to which said antibody or T-cell receptor binds. An
antigen may consist of numerous epitopes while a hapten, typically, may
possess few epitopes.

[0072] As used herein, the term "hapten" refers to a low-molecular weight
organic compound that is not capable of eliciting an immune response by
itself but will elicit an immune response once attached to a carrier
molecule.

3. Pegtide Dendrons

[0073] The present invention provides a carrier for the in vivo delivery
of a therapeutic agent according to Formula 1:

##STR00007##

[0074] wherein R is:

##STR00008##

[0075] R1 is:

##STR00009##

[0076] R2 is:

##STR00010##

[0077] PEG is linear or branched poly(ethylene glycol);

[0078] AA, AA1, AA2, and AA3 are each independently lysine
or ornithine;

[0083] In certain embodiments, Formula (I) may not contain PEG. In certain
other embodiments, R may not contain PEG, and Formula (I) may be
represented wherein R is:

##STR00012##

[0084] In certain embodiments, R may not contain Ld or Le. In
certain embodiments, R1 may not contain PEG. In certain embodiments,
R1 may not contain Lg or Lh. In certain embodiments,
R2 may not contain PEG. In certain embodiments, R2 may not
contain Lk or Lm.

[0085] One embodiment of the present invention includes a "third
generation" (G3.0) dendron wherein R2 is selected from the group
consisting of T1 and T2.

Another embodiment includes a "fourth generation" (G4.0) dendron in which
R2 is:

[0088] T1 and T2 are each a final PEG terminus selected from an
amino group, acetyl group, fluorenylmethyloxycarbonyl group; therapeutic,
diagnostic, biologic and targeting agents, as well as adjuvants, or any
combination thereof.

[0090] In one embodiment, the polyethylene glycol has a molecular weight
between about 100 and about 1000 Da. In another embodiment, the
polyethylene glycol has a molecular weight between about 250 and about
750 Da, In yet another embodiment, the polyethylene glycol has a
molecular weight between about 400 and about 600 Da.

[0091] Dendrons can be further joined together directly or through a
multifunctional core (convergent approach).

[0092] Dendrons may contain monodisperse PEG alternating with dipeptide
K-β-A, which leads to the formation of a monodisperse dendritic
structure with no structural heterogeneity.

[0093] It is possible to develop dendrons with different structure,
including sizes and densities, and multiple functionalities by altering
the monodisperse PEG and amino acids incorporated in dendrons. For
example, it is possible to incorporate amino acids like arginine and
histidine to complex the dendron with antisense oligonucleotides/siRNA as
illustrated in FIG. 18.

[0094] One of ordinary skill in the art will appreciate the various
embodiments that may be synthesized, and used in the presently described
and claimed invention.

4. Pharmaceutical Compositions

[0095] The present invention provides a pharmaceutical invention
comprising the PEGtide dendrons of the present invention and a
therapeutic agent, and may include a pharmaceutically acceptable carrier,
suitable for administration to a mammal, fish, bird, preferably a human.
To administer the pharmaceutical composition to humans or animals, it is
preferable to formulate the molecules in a composition comprising one or
more pharmaceutically acceptable carriers. The phrase "pharmaceutically
acceptable" refers to molecular entities and compositions that do not
produce allergic, or other adverse reactions when administered using
routes well-known in the art. "Pharmaceutically acceptable carriers"
include any and all clinically useful solvents, dispersion media,
coatings, antibacterial and antifungal agents, isotonic and absorption
delaying agents and the like.

[0096] The loading of therapeutic agents is achieved by any or the
combination of following mechanisms: (i) physical encapsulation; (ii)
complexation/charge interactions; and (iii) covalent conjugation via
non-degradable or degradable bonds. Physical encapsulation is achieved by
mixing the therapeutics with the dendron in varying proportions in
solvent or solvent systems. Charged interactions involve the creation of
multiple charged centers with in the dendron or the dendron surfaces for
complexation with therapeutic agents containing opposite charges. For
example, multiple arginine moieties are incorporated in the dendron to
make it cationic, which then complexes the anionic biomolecules
(oligonucleotides like antisense, siRNA etc.) into nano-sized materials.
The therapeutic agents are also attached to PEGtide dendrons via
non-degradable and/or degradable covalent bonds. Covalent attachment is
achieved by incorporating mutually reactive functional groups on the
therapeutic agent and the dendron or by using homo- and/or
hetero-multifunctional cross linkers. The examples of degradable covalent
bonds include, but not limited to, enzyme-sensitive peptide linkers and
bonds; auto-degradable ester and thioester bonds; acid-sensitive bonds
like imines, hydrazones, carboxylic hydrazones, ketal, acetal,
cis-aconityl, and trityl bonds; hypoxia-sensitive linkers; and
self-immolative bonds. BIOCONJUGATE TECHNIQUES (Academic Press; 1st
edition, Greg T. Hermanson, 1996) describes techniques for modifying or
crosslinking of biomolecules.

[0097] The therapeutic agent is released from the dendron by following
mechanisms: (i) passive diffusion; (ii) degradation of protease-sensitive
amide bonds used in the dendron construct; and (iii) degradation of
covalent bond linking the therapeutic agent to the dendron.

[0099] Particulate systems are taken up by mononuclear phagocyte system
(MPS) leading to eventual loss of therapeutic agents. Grafting of PEGs on
the dendron surfaces diminishes the uptake of nanoparticulate systems by
MPS. The same route is used for developing macrophage-targeted therapies.
PEGtide dendrons are also used for passive targeting to tumors by the
enhanced permeability and retention (EPR) effect. Unlike the normal
tissue, the blood vessels in tumor tissues are leaky and therefore
extensive leakage of blood plasma content (macromolecules, nanoparticles,
lipidic particles) occurs into the tumor, due to its enhanced
permeability. Since the lymphatic drainage in tumor tissue is also
impaired, the leaked contents exhibit size-dependent retention in tumor
tissues. Another reason size is critical in many delivery applications is
because molecules with smaller size (<5 nm) are rapidly eliminated by
kidneys. The PEG molecules display large hydrodynamic radii (larger than
expected from its molecular weight) because 2-3 molecules of water
surround each ethylene glycol moiety in PEG. The PEGtide dendrons, in
range of 100-200 nm, could be suitable candidate for passive targeting to
tumors by enhanced permeability and retention (EPR) effect (Matsumura,
Y., Cancer Research 46, 6387-6392, (1986)). The passive tumor targeting
properties of PEGtide dendrons are controlled by varying the molecular
size of the PEG incorporated in the dendron.

[0101] The present invention provides for a pharmaceutical composition
comprising of multiple therapeutic agents, as well as various types of
therapeutic agents. For example, a pharmaceutical agent may comprise a
diagnostic agent and a pharmaceutically active agent bound to a PEGtide
dendreon. In another example, multiple types of agents may be bound to a
PEGtide dendreon, such as at least one pharmaceutically active agent, at
least one biologic agent, at least one diagnostic agent and at least one
targeting agent, or various combinations thereof.

[0108] The invention also relates to a pharmaceutical composition for the
treatment of a hyperproliferative disorder or inflammatory disease or an
autoimmune disease in a mammal which comprises a therapeutically
effective amount of a compound of the invention and a pharmaceutically
acceptable carrier. In one embodiment said pharmaceutical composition is
for the treatment of cancer, including (but not limited to) the
following: carcinoma, including that of the bladder, breast, colon,
kidney, liver, lung, ovary, pancreas, stomach, cervix, thyroid and skin;
including squamous cell carcinoma; hematopoietic tumors of lymphoid
lineage, including leukemia, acute lymphocytic leukemia, acute
lymphoblastic leukemia, B-cell lymphoma, T-cell lymphoma, Burkitt's
lymphoma; hematopoietic tumors of myeloid lineage, including acute and
chronic myelogenous leukemias and promyelocytic leukemia; tumors of
mesenchymal origin, including fibrosarcoma and rhabdomyoscarcoma; other
tumors, including melanoma, seminoma, tetratocarcinoma, neuroblastoma and
glioma; tumors of the central and peripheral nervous system, including
astrocytoma, neuroblastoma, glioma, and schwannomas; tumors of
mesenchymal origin, including fibrosarcoma, rhabdomyoscarama, and
osteosarcoma; and other tumors, including melanoma, xeroderma
pigmentosum, keratoactanthoma, seminoma, thyroid follicular cancer and
teratocarcinoma, and other cancers yet to be determined in which CD38 is
expressed predominantly. In a preferred embodiment, the pharmaceutical
compositions of the invention are used for the treatment of a cancer such
as non-Hodgkin's lymphoma, Hodgkin's lymphoma, hairy cell leukemia,
multiple myeloma, chronic lymphocytic leukemia, chronic myeloid leukemia,
acute myeloid leukemia, or acute lymphocytic leukemia, in which CD38 is
expressed, and other cancers yet to be determined in which CD38 is
expressed predominantly. In another embodiment, the pharmaceutical
composition of the invention can be used to treat autoimmune diseases,
such as systemic lupus erythematosus, rheumatoid arthritis, multiple
sclerosis, Crohn's diasease, ulcerative colitis, gastritis, Hashimoto's
thyroiditis, ankylosing spondylitis, hepatitis C-associated
cryoglobulinemic vasculitis, chronic focal encephalitis, bulbous
pemphigoid, hemophilia A, membranoproliferative glomerulnephritis,
Sjogren's syndrome, adult and juvenile dermatomyositis, adult
polymyositis, chronic urticaria, primary biliary cirrhosis, idiopathic
thrombocytopenic purpura, neuromyelitis optica, Graves' dysthyroid
disease, bullous pemphigoid, membranoproliferative glonerulonephritis,
Churg-Strauss syndrome, and asthma. In another embodiment, said
pharmaceutical composition relates to other disorders such as, for
example, graft rejections, such as renal transplant rejection, liver
transplant rejection, lung transplant rejection, cardiac transplant
rejection, and bone marrow transplant rejection; graft versus host
disease; viral infections, such as mV infection, HIV infection, AIDS,
etc.; and parasite infections, such as giardiasis, amoebiasis,
schistosomiasis, and others as determined by one of ordinary skill in the
art.

[0109] When the pharmaceutical composition of the present invention is
used as a medicament, the compound of the present invention is mixed with
a pharmaceutically acceptable carrier (excipient, binder, disintegrant,
corrigent, flavor, emulsifier, diluent, solubilizing agents and the like)
to give a pharmaceutical composition which can be orally or parenterally
administered. A pharmaceutical composition can be formulated by a general
method.

[0111] The dose of the pharmaceutical composition of the present invention
is determined according to the age, body weight, general health
condition, sex, diet, administration time, administration method,
clearance rate, and the level of disease for which patients are
undergoing treatments at that time, or further in consideration of other
factors. While the daily dose of the compound of the present invention
varies depending on the condition and body weight of patient, the kind of
the compound, administration route and the like, it is parenterally
administered at, for example, 0.01 to 100 mg/patient/day by subcutaneous,
intravenous, intramuscular, transdermal, transocular, transpulmonary or
bronchial, transnasal or rectal administration.

[0112] Oral dosage forms may include capsules, tablets, emulsions and
aqueous suspensions, dispersions, and solutions. In the case of tablets,
commonly used carriers include, but are not limited to, lactose and corn
starch. Lubricating agents, such as, but not limited to, magnesium
stearate, also are typically added. For oral administration in a capsule
form, useful diluents include, but are not limited to, lactose and dried
corn starch. When aqueous suspensions or emulsions are administered
orally, the active ingredient can be suspended or dissolved in an oily
phase combined with emulsifying or suspending agents. If desired, certain
sweetening, flavoring, or coloring agents can be added.

[0113] In particular examples, an oral dosage range is from about 1.0 to
about 100 mg/kg body weight administered orally in single or divided
doses, including from about 1.0 to about 50 mg/kg body weight, from about
1.0 to about 25 mg/kg body weight, from about 1.0 to about 10 mg/kg body
weight (assuming an average body weight of approximately 70 kg; values
adjusted accordingly for persons weighing more or less than average). For
oral administration, the compositions are, for example, provided in the
form of a tablet containing from about 50 to about 1000 mg of the active
ingredient, particularly about 75 mg, about 100 mg, about 200 mg, about
400 mg, about 500 mg, about 600 mg, about 750 mg, or about 1000 mg of the
active ingredient for the symptomatic adjustment of the dosage to the
subject being treated.

5. Vaccine

[0114] The composition comprising according to Formula I described above
can be used in a vaccine formulation to immunize an animal. Thus, this
invention also provides an immunogenic or antigenic composition (a
vaccine) that contains a pharmaceutically acceptable carrier and an
effective amount of a composition comprising Formula I described above
further comprising an antigen, immunogen, epitope or a hapten. The
pharmaceutically acceptable carriers used in the vaccine can be selected
on the basis of the mode and route of administration, and standard
pharmaceutical practice.

[0115] Examples of a pharmaceutically acceptable carrier include, but are
not limited to, biocompatible vehicles, adjuvants, additives, and
diluents to achieve a composition usable as a dosage form. Examples of
other carriers include colloidal silicon oxide, magnesium stearate,
cellulose, and sodium lauryl sulfate. Additional suitable pharmaceutical
carriers and diluents, as well as pharmaceutical necessities for their
use, are described in Remington's Pharmaceutical Sciences.

[0116] A vaccine formulation may be administered to a subject per se or in
the form of a pharmaceutical composition as previously described. A
vaccine containing a biologic agent (such as a hapten, antigen,
immunogen, eptope) and an adjuvant may be manufactured by means of
conventional mixing, dissolving, granulating, dragee-making, levigating,
emulsifying, encapsulating, entrapping or lyophilizing processes. A
vaccine may be formulated in conventional manner using one or more
physiologically acceptable carriers, diluents, excipients or auxiliaries
which facilitate processing of the antigens of the invention into
preparations which can be used pharmaceutically. Proper formulation is
dependent upon the route of administration chosen.

[0117] Examples of an adjuvant include a cholera toxin, Escherichia coli
heat-labile enterotoxin, liposome, unmethylated DNA (CpG) or any other
innate immune-stimulating complex. Various adjuvants that can be used to
further increase the immunological response depend on the host species
and include Freund's adjuvant (complete and incomplete), mineral gels
such as aluminum hydroxide, surface-active substances such as
lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions,
keyhole limpet hemocyanin, and dinitrophenol. Useful human adjuvants
include BCG (bacille Calmette-Guerin) and Corynebacterium parvum.

[0118] An effective amount refers to the amount of an active
compound/agent that is required to confer a therapeutic effect on a
treated subject. Effective doses will vary, as recognized by those
skilled in the art, depending on the types of conditions treated, route
of administration, excipient usage, and the possibility of co-usage with
other therapeutic treatment.

[0119] The amount of a composition administered depends, for example, on
the particular antigen in the vaccine, whether an adjuvant is
co-administered with the antigen, the type of adjuvant co-administered,
the mode and frequency of administration, and the desired effect (e.g.,
protection or treatment), as can be determined by one skilled in the art.
Determination of an effective amount of the vaccine formulation for
administration is well within the capabilities of those skilled in the
art, especially in light of the detailed disclosure provided herein. An
effective dose can be estimated initially from in vitro assays. For
example, a dose can be formulated in animal models to achieve an
induction of an immune response using techniques that are well known in
the art. One having ordinary skill in the art could readily optimize
administration to all animal species based on results described herein.
Dosage amount and interval may be adjusted individually. For example, the
vaccine formulations of the invention may be administered in about 1 to 3
doses for a 1-36 week period. Preferably, 1 or 2 doses are administered,
at intervals of about 3 weeks to about 4 months; and booster vaccinations
may be given periodically thereafter. Alternative protocols may be
appropriate for individual animals. A suitable dose is an amount of the
vaccine formulation that, when administered as described above, is
capable of raising an immune response in an immunized animal sufficient
to protect the animal from an infection for at least 4 to 12 months. In
general, the amount of the antigen present in a dose ranges from about 1
pg to about 100 mg per kg of host, typically from about 10 pg to about 1
mg, and preferably from about 100 pg to about 1 pg. Suitable dose range
will vary with the route of injection and the size of the patient, but
will typically range from about 0.1 mL to about 5 mL. Sera can be taken
from the subject for testing the immune response or antibody production
elicited by the composition against the antigen. Methods of assaying
antibodies against a specific antigen are well known in the art.
Additional boosters can be given as needed. By varying the amount of the
composition and frequency of administration, the protocol can be
optimized for eliciting a maximal production of the antibodies.

[0120] A vaccine of this invention can be administered parenterally,
orally, nasally, rectally, topically, or buccally. The term "parenteral"
includes subcutaneous, intracutaneous, intravenous, intramuscular,
intraarticular, intraarterial, intrasynovial, intrasternal, intrathecal,
intralesional, or intracranial injection, as well as any suitable
infusion technique.

[0121] A sterile injectable composition can be a solution or suspension in
a non-toxic parenterally acceptable diluent or solvent. Such solutions
include, but are not limited to, 1,3-butanediol, mannitol, water,
Ringer's solution, and isotonic sodium chloride solution. In addition,
fixed oils can be conventionally employed as a solvent or suspending
medium (e.g., synthetic mono- or diglycerides). Fatty acid, such as, but
not limited to, oleic acid and its glyceride derivatives, are useful in
the preparation of injectables, as are natural pharmaceutically
acceptable oils, such as, but not limited to, olive oil or castor oil,
polyoxyethylated versions thereof. These oil solutions or suspensions
also can contain a long chain alcohol diluent or dispersant such as, but
not limited to, carboxymethyl cellulose, or similar dispersing agents.
Other commonly used surfactants, such as, but not limited to, Tweens or
Spans or other similar emulsifying agents or bioavailability enhancers,
which are commonly used in the manufacture of pharmaceutically acceptable
solid, liquid, or other dosage forms also can be used for the purpose of
formulation.

[0122] A vaccine for oral administration can be any orally acceptable
dosage form including capsules, tablets, emulsions and aqueous
suspensions, dispersions, and solutions. In the case of tablets, commonly
used carriers include, but are not limited to, lactose and corn starch.
Lubricating agents, such as, but not limited to, magnesium stearate, also
are typically added. For oral administration in a capsule form, useful
diluents include, but are not limited to, lactose and dried corn starch.
When aqueous suspensions or emulsions are administered orally, the active
ingredient can be suspended or dissolved in an oily phase combined with
emulsifying or suspending agents. If desired, certain sweetening,
flavoring, or coloring agents can be added.

[0123] In certain embodiments the antigen can come from a disease-causing
microorganism or a parasite. For example, the antigen can be one from a
virus, e.g., a flu invariant helix to elicit flu immunity, especially
pan-flu immunity. Other influenza antigens can be used to elicit immunity
to various distinct influenza types or other antigen of viral origin.

[0125] In some embodiments, the epitope can be a portion of a cancer
antigen, such that antibodies against the epitope can raise specific
anti-cancer immunity. This will be particularly interesting in situations
where passive infusion of specific antibodies is known to be therapeutic
(as is the case with neurofibromatosis, a childhood cancer), or where
specific anti-tumor antibodies can bind to receptors present in certain
cancer tissues (e.g. breast) and inhibit cancer growth (e.g.
Trastuzumab/herceptin, broadly used in breast cancer treatment to block
neu/her receptors).

[0126] In a further embodiment, the vaccine can target the mannose
receptor of macrophages, by conjugating mannose to the dendron in
conjunction with a cancer antigen to stimulate the immune system
conferring specific anti-cancer immunity.

[0127] The terms cancer antigen and tumor antigen are used interchangeably
and refer to an antigen that is differentially expressed by cancer cells.
Cancer antigens can be exploited to differentially target an immune
response against cancer cells, and stimulate tumor-specific immune
responses. Certain cancer antigens are encoded, though not necessarily
expressed, by normal cells. Some of these antigens may be characterized
as normally silent (i.e., not expressed) in normal cells, those that are
expressed only at certain stages of differentiation, and those that are
temporally expressed (e.g., embryonic and fetal antigens). Other cancer
antigens can be encoded by mutant cellular genes such as, for example,
oncogenes (e.g., activated ras oncogene), suppressor genes (e.g., mutant
p53), or fusion proteins resulting from internal deletions or chromosomal
translocations. Still other cancer antigens can be encoded by viral genes
such as those carried by RNA and DNA tumor viruses.

[0133] (1) 100 mg of Fmoc-Cys(Trt)-Wang Resin (0.65 mmol/g) was weighed
and transferred into a PD-10 column. The resin was swollen in
N,N-dimethylformamide (DMF), dichloromethane (DCM) and DMF for 10 minutes
each.

[0134] (2) The resin was drained, 10 ml piperidine/DMF (1/4 of volume
ratio) was added, and resin was placed on shaker for 20 minutes.

[0135] (3) The resin was washed with DMF 6-times and a small amount of
resin was used for Kaiser test. Moved to next cycle if the color of resin
turned blue, otherwise repeated step (2).

[0136] (4) 3.1 mg of Fmoc-β-Ala-OH (0.01 mmol), 4.1 mg
1-hydroxybenzotrizole (HOBT) (0.03 mmol), and 15.6 mg
Benzotriazole-1-yl-oxy-tris-pyrrolidino-phosphonium hexafluorophosphate
(PyBOP) (0.03 mmol) were dissolved in 0.1 ml of DMF. The mixture in DMF
was added to the PD-10 column containing the resin, and then 5.0 μl
N,N-diisopropylethylamine (DIEA) (0.03 mmol) was added and the column was
placed on shaker for 4 hours.

[0137] (5) The resin was washed with DMF for 6-times and column was
drained followed by Kaiser test.

[0138] (6) 61.3 μl of acetic anhydride (0.65 mmol) and DIEA (32.2
μl, 0.195 mmol) were transferred to the column, and column was placed
on shaker for 2 hours.

[0139] (7) The resin was washed with DMF for 3-times and Kaiser test was
performed. Moved to next step if the resin was colorless, otherwise
repeated step (6).

[0140] (8) Repeated steps (2) and (3).

[0141] (9) 17.7 mg of Fmoc-Lys(Fmoc)-OH (0.03 mmol), 4.1 mg of HOBT (0.03
mmol), and 15.6 mg of PyBOP (0.03 mmol) were dissolved into 0.1 ml of DMF
solution. The DMF mixture was transferred to the column and 5.0 μl of
DIEA (0.03 mmol) was added followed by shaking for 4 hours.

[0142] (10) The resin was washed with DMF and Kaiser test was performed.
If the resin was colorless, continued to step (11); otherwise, repeated
step (9).

[0143] (11) Repeated steps (2) and (3).

[0144] (12) 18.7 mg of Fmoc-β-Ala-OH (0.06 mmol), 8.1 mg of HOBT
(0.06 mmol), and 31.2 mg of PyBOP (0.06 mmol) were dissolved into 0.1 ml
of DMF solution. The DMF solution was transferred to the reaction column
and 9.9 μl of DIEA (0.06 mmol) was added followed by continuous
shaking for 4 hours.

[0145] (13) The resin was washed with DMF and Kaiser test was performed.
If the resin was colorless, continued to step (14); otherwise, repeated
step (12).

[0148] (16) The resin was washed with DMF and Kaiser test was performed.
If the resin was colorless, continued to step (17); otherwise, repeated
step (15).

[0149] (17) Repeated steps (2) and (3).

[0150] (18) 35.4 mg of Fmoc-Lys(Fmoc)-OH (0.06 mmol), 8.1 mg of HOBT (0.06
mmol), and 31.2 mg of PyBOP (0.06 mmol) were dissolved into 0.1 ml of
DMF. The DMF solution was transferred to the reaction column and 9.9
μl of DIEA (0.06 mmol) was added, followed by shaking for 4 hours.

[0151] (19) The resin was washed with DMF and Kaiser test was performed.
If the resin was colorless, continued to step (20); otherwise, repeated
step (18).

[0152] (20) Repeated steps (2) and (3).

[0153] (21) 37.4 mg of Fmoc-β-Ala-OH (0.12 mmol), 16.2 mg of HOBT
(0.12 mmol), and 62.4 mg of PyBOP (0.12 mmol) were dissolved into 0.1 ml
of DMF. The DMF solution was transferred to the reaction column and 19.8
μl of DIEA (0.12 mmol) was added followed by shaking for 4 hours.

[0154] (22) The resin was washed with DMF and Kaiser test was performed.
If the resin was colorless, continued to step (23); otherwise, repeated
step (21).

[0155] (23) Repeated steps (2) and (3).

[0156] (24) 100.8 mg of N-Fmoc-amide-dPEG12®-acid (Quanta
Biodesign Cat. No. 10283) (0.12 mmol), 16.2 mg of HOBT (0.12 mmol), and
62.4 mg of PyBOP (0.12 mmol) were dissolved into 0.1 ml of DMF. The DMF
solution was transferred to the reaction column and 19.8 μA of DIEA
(0.12 mmol) was added, followed by continued shaking for 4 hours.

[0157] (25) The resin was washed with DMF and Kaiser test was performed.
If the resin was colorless, continued to step (26); otherwise, repeated
step (24).

[0158] (26) Repeated steps (2) and (3).

[0159] (27) 70.9 mg of Fmoc-β-Ala-OH (0.12 mmol), 16.2 mg of HOBT
(0.12 mmol), and 62.4 mg of PyBOP (0.12 mmol) were dissolved into 0.1 ml
of DMF. The DMF solution was added to the reaction column and 19.8 μl
of DIEA (0.12 mmol) was added, followed by continued shaking for 4 hours.

[0160] (28) The resin was washed with DMF and Kaiser test was performed.
If the resin was colorless, continued to step (29); otherwise, repeated
step (27).

[0161] (29) Repeated steps (2) and (3).

[0162] (30) 74.7 mg of Fmoc-β-Ala-OH (0.24 mmol), 32.4 mg of HOBT
(0.24 mmol), and 124.9 mg of PyBOP (0.24 mmol) were dissolved into 0.1 ml
of DMF. The DMF solution was added to the reaction column and 39.7 μt
of DIEA (0.24 mmol) was added, followed by continued shaking for 4 hours.

[0163] (31) The resin was washed with DMF and Kaiser test was performed.
If the resin was colorless, continued to step (32); otherwise, repeated
step (30).

[0166] (34) The resin was washed with DMF and Kaiser test was performd. If
the resin was colorless, continued to step (35); otherwise, repeated step
(33).

[0167] (35) The resin was washed with DCM and dried under vacuum.

[0168] (36) The 10 ml of following cleavage cocktail solution was added to
the reaction column, followed by shaking for 2 hours. Trifluoroacetic
Acid (TFA)/1,2-Ethanedithiol/Water/Tri sopropylsi lane=94/2.5/2.5/1.

[0169] (37) The resin was filtered and the filtrate was collected,
concentrated, and precipitated from cold ether with stirring. The
precipitate was collected by centrifugation (3,000 rpm for 2 minutes).

[0170] (38) The precipitate was dissolved in DMF and step 37
(concentration followed by precipitation) was repeated 3-times.

[0171] (39) The precipitate was dried overnight under vacuum.

[0172] (40) MALDI-TOF analysis. The sample solution (1 mg/ml) was prepared
in aqueous solution containing 0.1% TFA. The 2 μl of sample solution
was taken and mixed with 21 μl of sinapinic acid matrix solution. The
1 μl of this mixture was spotted on the Opti-TOr® 384 Well Insert
Plate for MALDI-TOF (FIG. 2). The observed and calculated molecular
weights were 12240.3 D and 12258.5 D, respectively.

[0173] (41) DLS analysis

[0174] The sample solution (1 mg/ml) was prepared in water and used for
DLS studies. The radii of PEGTide dendron G3.0 was found as 53.9 nm (FIG.
3).

[0175] (1) 200 mg of Fmoc-β-Ala-Wang Resin (0.36 mmol/g) was weighed
and transferred into PD-10 column. The resin was swollen in. DMF two
times for 15 minutes.

[0176] (2) The resin was drained and then 10 ml of piperidine/DMF (1/4 of
volume ratio) was added, followed by shaking for 20 minutes.

[0177] (3) The resin was washed with DMF 4-times and a small amount of
resin was taken out for Kaiser test. If the color of the resin was blue,
continued to the next step; otherwise, repeated step (2).

[0178] (4) 14.5 mg of Fmoc-Lys (FAM)-OH (0.02 mmol), 10.8 mg of
1-hydroxybenzotrizole (HOBT) (0.08 mmol), and 41.6 mg of
benzotriazole-1-yl-oxy-tris-pyrrolidino-phosphonium hexafluorophosphate
(PyBOP) (0.08 mmol) were dissolved into 5.0 ml of DMF. The DMF solution
was added to the PD-10 column containing the resin and 26.4 μl of
N,N-diisopropyl-ethylamine (DIEA) (0.16 mmol) was added, and column was
placed on a shaker for 4 hours.

[0179] (5) The resin was washed with DMF 2-times and a small amount of
resin was taken out for Kaiser test. If the resin turned light blue,
moved to the next step.

[0180] (6) The 67.9 μl of acetic anhydride (0.72 mmol) and 35.7 μl
of DIEA (0.216 mmol) was added to the reaction column, followed by
shaking for 2 hours.

[0181] (7) The resin was washed with DMF 2-times and Kaiser test was
performed. If the resin was colorless, continued to next step; otherwise,
repeated the step (6).

[0182] (8) Repeated steps (2) and (3).

[0183] (9) 46.1 mg of N-Fmoc-amide-dPEG6®-acid (Quanta Biodesign
Cat. No. 10063) (0.08 mmol), 10.8 mg of HOBT (0.08 mmol), and 41.6 mg of
PyBOP (0.08 mmol) were dissolved into 5.0 ml of DMF. The DMF solution was
transferred to the reaction column and 26.4 μA of DIEA (0.16 mmol) was
added, followed by continued shaking for 4 hours.

[0184] (10) Repeated steps (7), (2) and (3)

[0185] (11) 47.3 mg of Fmoc-Lys(Fmoc)-OH (0.08 mmol), 10.8 mg of HOBT
(0.08 mmol), and 41.6 mg of PyBOP (0.08 mmol) were dissolved into 5.0 ml
of DMF. The DMF solution was transferred to the reaction column and 26.4
μl of DIEA (0.16 mmol) was added, followed by continued shaking for 4
hours.

[0186] (12) Repeated steps (7), (2) and (3)

[0187] (13) 49.8 mg of Fmoc-β-Ala-OH (0.16 mmol), 21.6 mg of HOBT
(0.16 mmol), and 83.3 mg of PyBOP (0.16 mmol) were dissolved into 5.0 ml
of DMF. The DMF solution was transferred to the reaction column and 52.9
μl of DIEA (0.32 mmol) was added, followed by continued shaking for 4
hours.

[0191] (17) 94.5 mg of Fmoc-Lys(Fmoc)-OH (0.16 mmol), 21.6 mg of HOBT
(0.16 mmol), and 83.3 mg of PyBOP (0.16 mmol) were dissolved into 5.0 ml
of DMF. The DMF solution was added to the reaction column and 52.9 μA
of DIEA (0.32 mmol) was added, followed by shaking for 4 hours.

[0192] (18) Repeated steps (7), (2) and (3)

[0193] (19) 99.6 mg of Fmoc-β-Ala-OH (0.32 mmol), 43.2 mg of HOBT
(0.32 mmol), and 166.5 mg of PyBOP (0.32 mmol) were dissolved into 5.0 ml
of DMF. The DMF solution was added to the reaction column and 105.8 μl
of DIEA (0.64 mmol) was added, followed by shaking for 4 hours.

[0194] (20) Repeated step (7).

[0195] (21) The resin was washed with DCM and dried under vacuum. The
resin containing 0.002 mmol of dendron was weighed and used for following
experiments.

[0196] (22) Repeated steps (2) and (3).

[0197] (23) 20.8 mg of Fmoc-Arg(Pbt)-OH (0.032 mmol), 4.4 mg of HOBT
(0.032 mmol), and 16.6 mg of PyBOP (0.032 mmol) were dissolved into 0.5
ml of DMF. The DMF solution was transferred to the reaction column and
10.6 μl of DIEA (0.064 mmol) was added, followed by shaking for 4
hours.

[0198] (24) Repeated steps (7), (2) and (3)

[0199] (25) Repeated step (23)

[0200] (26) Repeated steps (7), (2) and (3)

[0201] (27) 9.96 mg of Fmoc-β-Ala-OH (0.032 mmol), 4.32 mg of HOBT
(0.032 mmol), and 16.65 mg of PyBOP (0.032 mmol) were dissolved into 0.5
ml of DMF. The DMF solution was added to the reaction column and 10.6
μl of DIEA (0.064 mmol) was added and placed on shaker for overnight
period.

[0202] (28) Repeated steps (7), (2) and (3)

[0203] (29) Repeated step (23)

[0204] (30) Repeated steps (7), (2) and (3)

[0205] (31) Repeated step (27)

[0206] (32) Repeated steps (7), (2) and (3)

[0207] (33) 18.9 mg of Fmoc-Lys(Fmoc)-OH (0.032 mmol), 4.4 mg of HOBT
(0.032 mmol), and 16.6 mg of PyBOP (0.032 mmol) were dissolved into 0.5
ml of DMF. The DMF solution was added to the reaction column and 10.6
μl of DIEA (0.064 mmol) was added, followed by shaking for 4 hours.

[0208] (34) Repeated steps (7), (2) and (3)

[0209] (35) 19.9 mg of Fmoc-β-Ala-OH (0.064 mmol), 8.7 mg of HOBT
(0.064 mmol), and 33.3 mg of PyBOP (0.064 mmol) were dissolved into 0.5
ml of DMF. The DMF solution was added to the reaction column and 21.2
μl of DIEA (0.128 mmol) was added, followed by overnight shaking.

[0214] (40) The 10 ml of following cleavage cocktail solution was added
into the reaction column, followed by shaking for 2 hours.
Trifluoroacetic Acid (TFA)/Water/Triisopropylsilane=95/2.5/2.5.

[0215] (41) The resin was filtered and filtrate was collected, which was
concentrated and then precipitated from cold ether with stirring. The
precipitate was collected by centrifugation (3,000 rpm for 2 minutes).

[0216] (42) The precipitate was dissolved in DMF and reprecipitated from
cold ether as described in step (32). This process was repeated 3-times.

[0217] (43) The precipitate was dried under vacuum overnight.

[0218] (44) MALDI-TOF analysis"

[0219] The sample solution was prepared (1 mg/ml) in water containing 0.1
TFA %. The 2 μl of sample solution was mixed with 21 μl of
sinapinic acid matrix solution. 1 μl of this mixture was spotted on
the Opti-TOF® 384 Well Insert Plate for MALDI-TOF (FIG. 5). The
observed and calculated molecular weights were 8590.3 D and 8598.0 D,
respectively.

Example 1C

Synthesis of dv3-Containing Tetravalent PEGtide Dendron g2.0 (FIG. 6)

[0220] (1) Steps (1) to (20) described in EXAMPLE 1B were repeated.

[0221] (2) The resin was washed with DCM and dried under vacuum. The resin
containing 0.01 mmol of dendron was weighed and swollen with DMF 2-times
for 15 minutes each. Following experiments were carried out.

[0222] (3) The resin was drained and 10 ml of piperidine/DMF (1/4 of
volume ratio) was added, followed by shaking for 20 minutes.

[0223] (4) The resin was washed with DMF 4-times and a small amount of
resin was taken out for Kaiser test. If the color of the resin turned
blue, continued to the next step; otherwise, repeated step (3).

[0229] (10) 75.0 mg of Fmoc-Lys(Boc)-OH (0.16 mmol), 21.6 mg of HOBT (0.16
mmol), and 83.2 mg of PyBOP (0.16 mmol) were dissolved into 2.0 ml of
DMF. The DMF solution was transferred to the reaction column and 52.9
μl of DIEA (0.32 mmol) was added, followed by shaking for 4 hours.

[0230] (11) Repeated steps (6), (3) and (4).

[0231] (12) 65.8 mg of Fmoc-Asp(OtBu)-OH (0.16 mmol), 21.6 mg of HOBT
(0.16 mmol), and 83.2 mg of PyBOP (0.16 mmol) were dissolved into 2.0 ml
of DMF. The DMF solution was added to the reaction column and 52.9 μl
of DIEA (0.32 mmol) was added, followed by shaking for 4 hours.

[0232] (13) Repeated steps (6), (3) and (4).

[0233] (14) 54.0 mg of Fmoc-Pro-OH (0.16 mmol), 21.6 mg of HOBT (0.16
mmol), and 83.2 mg of PyBOP (0.16 mmol) were dissolved into 2.0 ml of
DMF. The DMF solution was transferred to the reaction column and 52.9
μl of DIEA (0.32 mmol) was added, followed by shaking for 4 hours.

[0234] (15) Repeated steps (6), (3) and (4).

[0235] (16) 103.8 mg of Fmoc-Arg(Pbf)-OH (0.16 mmol), 21.6 mg of HOBT
(0.16 mmol), and 83.2 mg of PyBOP (0.16 mmol) were dissolved into 2.0 ml
of DMF. The DMF solution was transferred to the reaction column and 52.9
μl of DIEA (0.32 mmol) was added, followed by shaking for 4 hours.

[0236] (17) Repeated steps (6), (3) and (4).

[0237] (18) 99.2 mg of Fmoc-His(Trt)-OH (0.16 mmol), 21.6 mg of HOBT (0.16
mmol), and 83.2 mg of PyBOP (0.16 mmol) were dissolved into 2.0 ml of
DMF. The DMF solution was transferred to the reaction column and 52.9
μl of DIEA (0.32 mmol) was added, followed by shaking for 4 hours.

[0238] (19) Repeated steps (6), (3) and (4).

[0239] (20) 84.3 mg of Fmoc-Trp(Boc)-OH (0.16 mmol), 21.6 mg of HOBT (0.16
mmol), and 83.2 mg of PyBOP (0.16 mmol) were dissolved into 2.0 ml of
DMF. The DMF solution was added to the reaction column and 52.9 μl of
DIEA (0.32 mmol) was added, followed by shaking for 4 hours.

[0240] (21) Repeated steps (6), (3) and (4).

[0241] (22) 61.3 mg of Fmoc-Ser(tBu)-OH (0.16 mmol), 21.6 mg of HOBT (0.16
mmol), and 83.2 mg of PyBOP (0.16 mmol) were dissolved into 2.0 ml of
DMF. The DMF solution was added to the reaction column and 52.9 μl of
DIEA (0.32 mmol) was added, followed by shaking for 4 hours.

[0242] (23) Repeated steps (6), (3) and (4).

[0243] (24) 49.8 mg of Fmoc-Ala-OH (0.16 mmol), 21.6 mg of HOBT (0.16
mmol), and 83.2 mg of PyBOP (0.16 mmol) were dissolved into 2.0 ml of
DMF. The DMF solution was transferred to the reaction column and 52.9
μl of DIEA (0.32 mmol) was added, followed by shaking for 4 hours.

[0244] (25) Repeated steps (6), (3) and (4).

[0245] (26) 47.6 mg of Fmoc-Gly-OH (0.16 mmol), 21.6 mg of HOBT (0.16
mmol), and 83.2 mg of PyBOP (0.16 mmol) were dissolved into 2.0 ml of
DMF. The DMF solution was transferred to the reaction column then 52.9
μl of DIEA (0.32 mmol) was added, followed by shaking for 4 hours.

[0246] (27) Repeated steps (6), (3) and (4).

[0247] (28) 56.5 mg of Fmoc-Leu-OH (0.16 mmol), 21.6 mg of HOBT (0.16
mmol), and 83.2 mg PyBOP (0.16 mmol) were dissolved into 2.0 ml of DMF
solution. The DMF solution was added to the reaction column and add 52.9
μl of DIEA (0.32 mmol) was added, followed by shaking for 4 hours.

[0248] (29) Repeated step (6), (3) and (4).

[0249] (30) The resin was washed with DCM and dried under vacuum.

[0250] (31) The 10 ml of following cleavage cocktail solution was added
into the reaction column, followed by shaking for 2 hours.
Trifluoroacetic Acid (TFA)/Water/Triisopropylsilane=95/2.5/2.5.

[0251] (32) The resin was filtered and filtrate was collected, which was
concentrated and precipitated from cold ether with stirring. The
precipitate was collected by centrifugation (3,000 rpm for 2 minutes).

[0252] (33) The precipitate was dissolve in DMF and re-precipitated as
described in step (32). This process was repeated 3-times.

[0253] (34) The precipitate was dried under vacuum overnight.

[0254] (35) MALDI-TOF analysis.

[0255] The sample solution was prepared (1 mg, m/1) in aqueous solution
containing 0.1 TFA %. The 2 μl of sample solution was taken and mixed
with 21 μl of sinapinic acid matrix solution. The 1 μl of this
mixture was spotted on the Opti-TOF® 384 Well Insert Plate for
MALDI-TOF (FIG. 7). The observed and calculated molecular weights were
8554.1 D and 8554.5 D, respectively.

Example 1D

Synthesis of A-K(5-FAM)-PEG6-Fmoc (1)

[0256] The derivative (1) was synthesized on Fmoc-β-Ala-Wang Resin
(loading capacity: 0.36 mmole/g) in following steps.

[0257] (1) 0.1 g of Wang resin was swollen in DMF for 1 hr and washed with
DMF (3×3 mL).

[0263] (7) The visual Kaiser test was used to monitor completion of each
deprotection and coupling steps (Kutscher, H. L., International Journal
of Pharmaceutics 402, 64-71, (2010)) few resin beads were taken out in a
vial and 2 drops of each ninhydrin, phenol and pyridine were added to it.
The vial was heated at 110° C. for 3 min. Finally the resin was
washed and dried in vacuum (108.1 mg resin was obtained, 8.1 mg mass
gain: 98.6%). MALDI-TOF: MW: calcd. 1132.45 Da, found 1130.38 Da.

[0265] G1.0. To obtain PEGtide dendron G1.0, three cycles of step 2, 3,
and 4 (described above) were repeated to couple Fmoc-Lys(Fmoc)-OH (23.6
mg, 0.04 mmole), Fmoc-β-Ala-OH (24.9 mg, 0.08 mmole), and
Fmoc-PEG6-OH (46.1 mg, 0.08 mmole), respectively. About 4 equiv. of
PyBOP and HOBT, and 8 equiv. of DIPEA were used in each coupling step. At
the end of these coupling cycles, step 2 was repeated one more time to
remove the terminal Fmoc groups. The visual Kaiser test was used to
monitor completion of each deprotection and coupling steps (step 7). The
resin was washed with DMF (2×3 mL), DCM (2×3 mL), and
methanol (2×3 mL) and dried overnight under vacuum to obtain the
resin-bound PEGtide dendrons G1.0 (Yield. 118.1 mg, 18.1 mg mass gain,
91%). Finally, the PEGtides were cleaved from support by treating with a
cocktail of TFA/water/triisopropylsilane (TIS) (95/2.5/2.5 v/v/v) for 2
hrs. The cleavage cocktail was removed under vacuum, and the crude
dendrons were stored at -20° C. and further purified by HPLC.
MALDI-TOF: MW, calcd. 1851.68 Da, found 1849.72 Da.

[0268] PEGtide dendrons of different generations, G1.0-4.0, were
synthesized by sequentially assembling Fmoc-K(Fmoc)-OH, Fmoc-β-A-OH,
and Fmoc-PEG6-OH on a solid support. The Fmoc approach of
solid-phase peptide synthesis (SPPS) and a divergent strategy was used to
obtain PEGtide dendrons, G1.0-4.0. Briefly, Fmoc-K(5-FAM)-OH was coupled
to Fmoc-β-A-Wang resin to obtain resin-bound
A-K(5-FAM)-PEG6-Fmoc (1) (FIG. 9A). The 5-FAM-labeled lysine (K) was
incorporated to detect dendrons in biological assays. The synthesis of
PEGtide dendron G1.0 was initiated by coupling Fmoc-K(Fmoc)-OH to (1). In
the next step, Fmoc-β-A-OH was coupled to α-ε-amino
groups of lysine. Finally, monodisperse Fmoc-PEG6-OH was coupled to
both alanine (A) moieties to obtain resin-bound G1.0 dendrons (2). The
G1.0 dendrons were obtained after deprotection of terminal Fmoc groups
and cleavage from support. Usually large excess of amino acid/PEG was
used to drive reaction to completion, and HOBT and PyBOP were used for
coupling due to their high reactivity and chiral stability of
benzotriazoyl esters of amino acid/peptide. Relatively longer durations
were used for coupling and deprotection reactions.

[0269] The next generation dendrons were synthesized from resin-bound
dendrons of previous generation using a procedure described above for the
synthesis of G1.0 (FIG. 9B). For example, the dendrons G2.0 were obtained
by sequentially coupling Fmoc-K(Fmoc)-OH, Fmoc-β-A-OH, and
Fmoc-PEG6-OH to resin-bound dendron G1.0 (2). Similarly, the PEGtide
dendrons G3.0 and 4.0 were obtained from resin-bound dendrons G2.0 (3)
and G3.0 (4), respectively. Thus, each PEGtide dendron contains
monodisperse PEG (MW: 565.65 Da) interspersed with dipeptide
lysine-alanine (-K-β-A-). The number of free terminal amino groups
in G1.0, 2.0, 3.0, and 4.0 are 2, 4, 8, and 16, respectively that can be
utilized for further modification with drugs, targeting groups, and
imaging moieties.

[0270] All dendrons were purified by HPLC, and characterized using
MALDI-TOF and DLS. The purity of PEGtide dendrons was determined using
HPLC equipped with fluorescence detector. A fluorescence detector (Ex/Em:
492/518 nm) was employed because the first lysine incorporated in the
dendron core was labeled with 5-FAM. The retention times of PEGtide
dendron, G1.0-4.0, were found as 33.48, 35.05, 37.96, and 39.37 min,
respectively. Thus, the retention time increased with increase in
generation of dendrons because the avidity of binding to the column
increases with contact area between the surfaces, as well as the binding
strength of each interaction. Similar behavior has been reported for
amine terminated PAMAM dendrons. The HPLC profiles also demonstrated that
dendrons were obtained in high purity. The percentage purities of
G1.0-4.0 dendrons were found as 98.3, 99.0, 96.8, and 98.9, respectively.

[0271] The molecular weights of PEGtide dendron G1.0-4.0 were determined
using MALDI-TOF. The molecular weights of G1.0, G2.0, G3.0, and G4.0 were
estimated as 1849.72, 3730.31, 7486.30, and 15022.1 Da, respectively,
which were in agreement with calculated values of 1851.68, 3733.06,
7497.30, and 15023.77 Da. A representative MALD1-TOF spectrum of purified
PEGtide dendron G4.0 is shown in FIG. 10. The signal intensity was
relatively low when compared to spectra of low generation dendrons
(G1.0-3.0). This is because larger molecule require more laser power for
desorption and ionization process. Nevertheless, the ratio of signal to
noise was still higher than 10. It must be mentioned that in each case
single molecular weight peak was obtained, which is not characteristics
of PEG polymers. This single molecular weight peak is due to the use of
monodisperse PEG in dendron structure.

[0272] The hydrodynamic radii of PEGtide dendrons, G1.0-4.0, were
determined using DLS. The radii for G1.0, G2.0, G3.0, and G4.0 dendrons
were determined as 28.2, 65.4, 86.4, 114.3 nm. Thus, hydrodynamic radii
increased with increase in generation of dendrons, which was not
unexpected because hydrodynamic radii of peptides, proteins, and polymers
are known to increase with increasing molecular weights. The narrow size
distribution of dendrons points to the advantage of using mono-disperse
PEG in dendron structure.

[0275] To obtain dendrons containing octavalent mannose cluster,
α-D-mannopyranosylphenyl isothiocyanate was coupled to PEGtide G3.0
at room temperature in buffer (pH, 9.0) (as illustrated in FIG. 13). The
coupling reaction used here involves the attack of nucleophile on the
central electrophilic carbon of the isothiocynate group, leading to the
formation of thiourea (FIG. 12). The G3.0-Mannose was purified by
dialysis (MWCO: 3000 Da) in water. Despite the dialysis, the HPLC profile
of the conjugate showed presence of several impurities, possibly due to
the coupling of varying number of mannose moieties to G3.0 dendron.
Therefore, G3.0-Mannose was further purified using HPLC, and fraction
corresponding to retention time of 75.3 min was collected. The molecular
weight of purified conjugate was estimated as 10009.9 Da using MALDI-TOF,
which was in agreement with calculated value of 10003.9 Da, corresponding
to attachment of eight mannose moieties onto the dendron (as illustrated
in FIG. 14). The hydrodynamic radius of conjugates was estimated as 90.6
nm by DLS (as illustrated in FIG. 15), which was only slightly higher
than PEGtide G3.0, with hydrodynamic radius of 86.4 nm.

[0315] (1) Synthesis PEGtide dendron G2.0 according to steps from (1) to
(20) in EXAMPLE 1F.

[0316] (2) The resin was washed by DMF solvent (4 mL×3) and
monitored by Kaiser Test. Moved to next step if the small part of resin
showed transparent after Kaiser Test, otherwise repeated last one
coupling step.

[0317] (3) 4 mL of piperidine in DMF (1:4 of volume ratio) was added to
resin for continually shaking 20 minutes then drained.

[0318] (4) The resin was washed by DMF solvent (4 mL×3) and
monitored by Kaiser Test. Moved to next cycle if the small part of resin
showed blue after Kaiser Test, otherwise repeated step (3).

[0337] Using MALDI-TOF, purified DV3-G2.0 collected from semi-preparative
HPLC purification, the calculated and found molecular weights are 8554.5
Da and 8547.6 Da, respectively.

Example 2

Macrophage Uptake of Dendrons

[0338] J774.E murine macrophage cells were grown as attached cells in a
CO2 water-jacketed incubator (Form a Scientific Inc., Marietta,
Ohio) with a humidified 5% CO2 atmosphere at 37° C. The cells
were maintained in RPMI-1640 (GIBCO) containing 10% fetal bovine serum
(FBS), 100 U/mL penicillin, and 100 μg/mL streptomycin/mL. The cells
were seeded two days before experiment to 24-well plates at
0.5×105 cells/well for quantitative uptake measurement or to
chambered coverglass (LAB-TEK II) at 1×105 cell/chamber for
confocal microscopy. Cells were incubated with G3.0-Mannose (40 nM) in
HBSS at 37° C. for 1 hr. For confocal microscopy, the incubation
included the additional 0.16 mg/mL of the general endocytosis marker
rhodamine B-labeled dextran (10,000 MW) and the nuclear dye DAPI. For
Mannan inhibition studies, cells were pre-incubated with mannan (10
mg/mL) for 1 hr, followed by incubation with G3.0-Mannose, with or
without rhodamine B-dextran/DAPI plus 10 mg/mL of mannan. After
incubation, the unbound molecules were washed thrice using cold DPBS
buffer. PEGtide dendron G3.0 (40 nM) (without mannose) was used as a
negative control of mannose receptor-mediated cellular uptake.

[0339] The buffer was collected after 1 hr incubation and filtered through
a Microcon (NMWL: 3 kDa) to demonstrate that no detectable free
fluorescence had been produced during the 1 hr incubation time. In
quantitative uptake experiments, the washed cells were lysed in wells
overnight with 150 μL of 1 N NaOH solution and neutralized the next
day with 150 μL of 1 N HCl solution, resulting in 300 μL of cell
lysate/well. 250 μL of cell lysate was transferred to a well of a
96-well plate for 5-FAM fluorescence measurement on Tecan GENIOS
fluorescent plate reader (Phenix Research Products, Hayward, Calif.). The
total cell-associated fluorescence reading was converted into total
cell-associated dendron amount using a separately established dendron's
fluorescence standard curve. A small fraction of the lysate (about 50
μL, depending on cell density plated) was used to determine total
cellular protein/well by Bradford Protein Assay. The total cellular
protein/well was used to normaliie the dendron amount/well to correct
well-to-well variation in cell mass. To demonstrate that the total
cell-associated dendron amount was mostly due to internalized
fluorescence, as opposed to cell-surface-bound fluorescence, confocal
microscopy was carried out in a Z-stack scanning mode on a Leica TCS SP5
confocal microscope (Leica Microsystems Inc., Buffalo Grove, Ill.).

[0340] The quantitative uptake of G3.0-Mannose in macrophage was
699.6±1.8 pmole/μg, whereas that of non-mannosylted G3.0 (control)
was 13.1±4.7 pmole/μg, indicating a 53-fold specificity for mannose
receptor. The mannose analog mannan inhibited the G3.0-Mannose uptake to
7.5±4.7 pmole/μg, further suggesting that the uptake was mannose
receptor-mediated (FIG. 16).

[0341] To demonstrate that the uptake of total cellular dendron amount is
mostly intracellular, the confocal microscopy was carried out. Punctuate
red fluorescence of the fluid endocytosis marker rhodamine B-labeled
dextran was significant in both treatments, indicating that the general
fluid endocytosis was normal in both experimental settings, regardless of
the expected engagement of mannose receptor by G3.0-Mannose.

[0342] In contrast, the intracellular green fluorescence from 5-FAM
conjugated G3.0-Mannose was prominent only in G3.0-Mannose treated cells
but not in G3.0 treated cells, suggesting that G3.0-Mannose was taken
into cells in a mannose receptor-dependent manner. Since little cell
surface green fluorescence was present, the images suggested little cell
surface binding and the total cellular associated G3.0-Mannose reflected
mostly internalized G3.0-Mannose dendrons. The punctuate appearance of
the green fluorescence and its co-localization with the red fluid
endocytosis marker suggested that G3.0-Mannose was internalized through
endosytosis. As expected, all punctuate green fluorescence merged with
punctuate red fluorescence but not vice versa, suggesting that mannose
receptor-mediated G3.0-Mannose endocytosis uses only one of the several
occurring endocytosis pathways.

[0353] The specification is most thoroughly understood in light of the
teachings of the references cited within the specification. The
embodiments within the specification provide an illustration of
embodiments of the invention and should not be construed to limit the
scope of the invention. The skilled artisan readily recognizes that many
other embodiments are encompassed by the invention. All publications, and
patent applications cited in this disclosure are incorporated by
reference in their entirety. To the extent the material incorporated by
reference contradicts or is inconsistent with this specification, the
specification will supersede any such material. The citation of any
references herein is not an admission that such references are prior art
to the present invention.

[0354] Those skilled in the art will recognize, or be able to ascertain
using no more than routine experimentation, many equivalents to the
specific embodiments of the invention described herein. Such equivalents
are intended to be encompassed by the following embodiments.